Method of producing non-bovine chymosin and use hereof

ABSTRACT

A method of recombinantly producing a non-bovine pre-prochymosin, prochymosin or chymosin derived from ruminant species including deer species, buffalo species, antelope species, giraffe species, ovine species and caprine species; Camelidae species such as  Camelus dromedarius ; porcine species; or Equidae species. The recombinant enzymes are used in milk coagulating compositions in cheese manufacturing based on cow&#39;s milk and milk from any animal species which are used in cheese manufacturing including camel&#39;s milk.

This is a continuation of Ser. No. 09/985,936 filed Nov. 6, 2001, nowabandoned; which is a continuation-in-part of 09/705,917 filed Nov. 6,2000, now abandoned.

The prior application(s) set forth above are hereby incorporated byreference in their entirety.

FIELD OF INVENTION

The present invention relates generally to the field of cheesemanufacturing. In particular, novel recombinant means of providingmilk-clotting enzymes of non-bovine, ie non-Bos taurus, animal originare provided. Specifically, the invention pertains to a process ofrecombinantly providing pre-prochymosin, prochymosin and chymosin ofnon-bovine origin including such enzymes that are derived from camels.

TECHNICAL BACKGROUND AND PRIOR ART

Enzymatic coagulation of milk-by-milk clotting enzymes, such as chymosinand pepsin, is one of the most important processes in the manufacture ofcheeses. Enzymatic milk coagulation is a two-phase process: a firstphase where a proteolytic enzyme, chymosin or pepsin, attacks κ-casein,resulting in a metastable state of the casein micelle structure and asecond phase, where the milk subsequently coagulates and forms acoagulum.

Chymosin (EC 3.4.23.4) and pepsin (EC 3.4.23.1), the milk clottingenzymes of the mammalian stomach, are aspartic proteases belonging to abroad class of peptidases (Kappeler, 1998). Aspartic proteases are foundin eukaryotes, retroviruses and some plant viruses. Eukaryotic asparticproteases are monomers of about 35 kDa, which are folded into a pair oftandemly arranged domains with a high degree of similarity, i.e. 20% orhigher. The overall secondary structure consists almost entirely ofpleated sheets and is low in α-helices. Each domain contains an activesite centred on a catalytic aspartyl residue with a consensus sequencehydrophobic-Asp-Thr-Gly-Ser/Thr (SEQ ID NO:7) which aids in maintainingthe correct Φ-loop conformation of the site, and with multiplehydrophobic residues near the aspartic residue. The two catalytic sitesare arranged face-to-face in the tertiary structure of correctly foldedproteins. In bovine chymosin, the distance between the aspartic sidechains is about 3.5 Å. The residues are reported to be extensivelyhydrogen bonded, concomitantly with the adjacent threonine residues, tothe corresponding residues of the other domain or the neighbouring atomsof the own domain, to stabilise the correct position. Optimum activityof an aspartic protease is achieved when one of the aspartic residues isprotonated and the other one is negatively charged. The active sites ofchymosin and other aspartic proteases are embedded, with lowaccessibility, in the middle of a cleft, about 40 Å in length, whichseparates the two domains, and which is covered by a flap that, inbovine and camel chymosin, extends from about Leu73 to Ile85 in theN-terminal domain.

When produced in the gastric mucosal cells, chymosin and pepsin occur asenzymatically inactive pre-prochymosin and pre-pepsinogen, respectively.When chymosin is excreted, an N-terminal peptide fragment, thepre-fragment (signal peptide) is cleaved off to give prochymosinincluding a pro-fragment. Prochymosin is a substantially inactive formof the enzyme which, however, becomes activated under acidic conditionsto the active chymosin by autocatalytic removal of the pro-fragment.This activation occurs in vivo in the gastric lumen under appropriate pHconditions or in vitro under acidic conditions.

The structural and functional characteristics of bovine, ie Bos taurus,pre-prochymosin, prochymosin and chymosin have been studied extensively(Foltman et al. 1977). The pre-part of the bovine pre-prochymosinmolecule comprises 16 aa residues and the pro-part of the correspondingprochymosin has a length of 42 aa residues. Foltman et al., 1997 haveshown that the active bovine chymosin comprising 323 aa is a mixture oftwo forms, A and B, both of which are active, and sequencing dataindicate that the only difference between those two forms is anaspartate residue at position 290 in chymosin A and a glycine residue atthat position in chymosin B.

Whereas chymosin is produced naturally in mammalian species includingruminant species such as bovines, caprines, buffaloes and ovines; pigs(Houen et al., 1996); Camelidae species; primates including humans andmonkeys; and rats, bovine chymosin and (to a lesser extent) caprinechymosin are presently the only of these animal chymosin species thatare commercially available to the dairy industry. Bovine chymosin, inparticular calf chymosin, is commercially available both as stomachenzyme extracts (rennets) comprising the natively produced chymosin andas recombinantly produced chymosin which is expressed in bacterial,yeast or fungal host cells (see e.g. WO 95/29999, Ward et al. 1990).

Recently, studies on functional characteristics of rennet extracted fromthe stomach of Camelus dromedarius chymosin have been reported (Wangohet al., 1993; Elagamy, 2000) and it has been found that the clottingtime of camel's milk is significantly reduced when camel rennet is usedinstead of bovine calf rennet. Fractions of crude camel and calfrennets, which were isolated by anion-exchange chromatography, have beentested for their respective capabilities to clot camel's milk and cow'smilk and it was found that the main clotting activity of calf rennet(i.e. an extract containing both chymosin and pepsin) resides in thepepsin fraction, i.e. bovine chymosin is substantially inactive inrespect of clotting camel's milk, whereas the main clotting activity ofcamel rennet extracts on camel's milk resided in a first fraction that,compared to calf chymosin, eluted at a somewhat lower NaClconcentration. The active enzyme of this fraction has not yet beencharacterised, but it is assumingly chymosin. It has also beendemonstrated that this camel rennet fraction has a clotting activity oncow's milk that is similar to that of bovine chymosin (Wangoh et al.,1993). It is evident, therefore, that more effective clotting of camel'smilk could be achieved at an industrial level were camel chymosincommercially available and it is also conceivable that camel chymosin ishighly suitable as a cow's milk clotting enzyme as well.

The primary structure of chymosin isolated from gastric mucosa of camelshas been determined (Kappeler, 1998). The mature and active form ofcamel chymosin is 323 aa residues long and it has a molecular weight of35.6 kDa and an isoelectric point at pH 4.71. It shows 85.1% aa sequenceidentity with bovine chymosin.

Presently, bovine chymosin is manufactured industrially usingrecombinant DNA technology, e.g. using filamentous fungi such asAspergillus species (see e.g. Ward, 1990), yeast strains, e.g. ofKlyuveromyces species, or bacterial species, e.g. E. coli, as hostorganisms. Such recombinant microbial production strains are constructedand continuously improved using DNA technology as well as classicalstrain improvement measures directed towards optimising the expressionand secretion of the heterologous protein, but it is evident that theproductivity in terms of overall yield of gene product is an importantfactor for the cost effectiveness of industrial production of theenzyme. Accordingly, a continued industrial need exists to improve theyield of chymosin in recombinant expression systems.

Whereas efforts to improve yields of chymosin activity up till now haveexclusively been concerned with chymosin of bovine origin, the industryhas not yet explored the possibility of providing effective chymosinpreparations based on non-bovine, ie non-Bos taurus, chymosin species.However, the present inventors have surprisingly found that it ispossible to provide industrially highly useful non-bovine chymosin usingrecombinant DNA technology at a production yield level which, relativeto that which can be obtained in current, optimised bovine chymosinproduction systems, is significantly improved.

In addition to the potential of significantly improved chymosinproduction cost-effectiveness, the provision of such non-bovine chymosinspecies at a commercial level makes available chymosin products that arenot only capable of clotting cow's milk at least as effectively aschymosin of bovine origin, but which, additionally, are capable of moreeffectively clotting milk from other animal species including milk ofthe source species. Specifically, the invention has made it possible toprovide, for the first time, camel chymosin in sufficient quantities torender an industrial, cost-effective and high quality production ofcheese based on camel's milk possible, which, due to the scarcity ofcamel calf stomach material, has not hitherto been possible.

Additionally, it has been discovered that camel chymosin has a highclotting activity on cow's milk, which renders the enzyme useful formanufacturing cheese based on cow's milk. It was a surprising finding ofthe present inventors that camel chymosin has a specific κ-caseinhydrolysing activity (Phe-Met 105/106), i.e. C/P ratio as definedhereinbelow, which is superior to that of bovine chymosin. A higher C/Pratio implies generally that the loss of protein during cheesemanufacturing due to non-specific protein degradation is reduced, i.e.the yield of cheese is improved, and that the development of bittertaste in the cheese during maturation is reduced.

SUMMARY OF THE INVENTION

The invention relates in one aspect to a method of producing anon-bovine pre-prochymosin, prochymosin or chymosin, the methodcomprising the steps of (i) isolating or constructing a nucleic acidsequence coding for the pre-prochymosin, prochymosin or chymosin, (ii)constructing an expression vector comprising said coding sequence and,operably linked thereto, appropriate expression signals permitting thepre-prochymosin, prochymosin or chymosin to be expressed in a host cell,(iii) transforming said host cell with the expression vector, (iv)cultivating the thus transformed host cell under conditions where thecoding sequence is expressed and (v) harvesting the pre-prochymosin,prochymosin or chymosin. As used herein the expression “non-bovinepre-prochymosin, prochymosin or chymosin” refers to such enzymes orprecursors herefor that are derived from a mammalian species other thanBos taurus.

In further aspects, the invention pertains to a DNA construct capable ofexpressing non-bovine pre-prochymosin, prochymosin or chymosin, saidconstruct comprising an expression vector comprising a nucleic acidsequence comprising a gene coding for the pre-prochymosin, prochymosinor chymosin and, operably linked thereto, appropriate expression signalspermitting the pre-prochymosin, prochymosin or chymosin to be expressedin a host cell, and to a host cell transformed with such a DNAconstruct.

In still further aspects a composition is provided comprising anon-bovine pre-prochymosin, prochymosin or chymosin produced by theabove method including such an enzyme that is in a substantiallydeglycosylated form and a method of manufacturing cheese, comprisingadding a milk clotting effective amount of such a composition to milkand carrying out appropriate further cheese manufacturing steps.

In yet another aspect, the invention relates to a method ofmanufacturing cheese, comprising adding a milk clotting effective amountof a non-bovine prochymosin or chymosin to the milk and carrying outappropriate further cheese manufacturing steps, the non-bovineprochymosin or chymosin having in said milk a C/P ratio as determinedherein which is in the range of 2-20.

In other aspects the invention provides a milk clotting compositioncomprising a bovine milk clotting enzyme selected from prochymosin,chymosin and pepsin and a non-bovine milk clotting enzyme selected fromprochymosin, chymosin, pepsin and a microbial aspartic protease and amethod of manufacturing cheese from milk, comprising adding to milk amilk clotting effective amount of such a composition, and carrying outappropriate further cheese manufacturing steps.

DETAILED DISCLOSURE OF THE INVENTION

In accordance with the invention, there is, in one aspect of theinvention, provided a method of recombinantly producing pre-prochymosin,prochymosin or chymosin of non-bovine origin.

For the purposes of this application, the expression “non-bovine origin”refers to any non-Bos taurus mammalian species where pre-prochymosin isproduced naturally in the gastrointestinal tract. Such species includeany of those mentioned above, e.g. ovine species, caprine species andCamelidae species comprising the genus Camelus with the two speciesCamelus dromedarius and Camelus bactrianus; buffalo species includingwater buffaloes, Indian buffaloes and Cape buffaloes, the genus Lamaincluding Lama glama, Lama guanicoe and Lama paco; and the genusVicugna. Camels are ruminating, but do not belong to the suborderRuminantia as do e.g. bovine, ovine and caprine species, but they belongto the suborder Tylopoda.

However, a non-bovine chymosin as used in this context may also includea chymosin molecule encoded by a cluster or a shuffling of DNA segmentsof different origin resulting in complex rearrangements of the DNA.Shuffling of DNA segments or gene shuffling is in the present inventionin general to be construed as a method for the construction of chimericgenes resulting in genes coding for chimeric proteins. Such proteinswill consist of domains derived from two or more parental proteins. Thechimeric genes may be constructed either on the basis of rational designbased on knowledge of protein function or on the basis of combinatoriallaboratory methods generating random chimeric genes. Such randomcombinatorial libraries can be screened for the identification ofoptimal enzymes by a variety of screening procedures.

Prochymosin is in the present context to be understood as the precurseror proenzyme of chymosin. Prochymosin appears to possess a basic leadersequence (pro-part) on the N-terminal side of chymosin and said leadersequence is believed to be cleaved off during activation of theprochymosin. Furthermore in this context preprochymosin consists ofprochymosin to which is added on the N-terminal end of prochymosin ahydrophobic leader sequence. This leader sequence, also called secretionsignal or prepart, is cleaved off when the protein is secreted. Chymosinis in the cell initially synthesised as pre-prochymosin. (Harris, T. J.,Lowe, P. A., Lyons, A., Thomas, P. G., Millican, T. A., Ptael, T. P.,Bose, C. C., Carey, N. H., Doel, M. T. Nucleic acid Research 1982, Apr.10, 2177-2187 Molecular cloning and nucleotide sequence of cDNA codingfor calf preprochymosin.)

In an initial step of this method, a nucleic acid sequence, i.e. apolynucleotide, of non-bovine origin that codes for pre-prochymosin,prochymosin or chymosin is provided. The skilled artisan will appreciatethat several approaches for obtaining such a sequence can be usedincluding one based on the isolation of mRNA from mucosal cells of theselected source animal species and using this RNA as template in anucleotide amplification procedure such as a PCR reaction using suitablesense and anti-sense primers which e.g. may be constructed syntheticallybased on the known sequences for bovine chymosin species. The person ofskill in the art will appreciate that other methods for obtaining acoding sequence according to the invention may be used such ashybridisation procedures using as probes fragments of known codingsequences for chymosin that will permit the presence of homologous DNAor RNA to be detected in preparations of cells of the selectednon-bovine source species. Alternatively, it is possible to construct acoding sequence based on the isolation of the non-bovinepre-prochymosin, prochymosin or chymosin followed by determining theamino acid sequence of the enzyme or fragments hereof which in turnpermits the construction of primer oligonucleotides for detection andconstruction of coding sequences. The basic techniques that are requiredin the above procedures of obtaining coding sequences are generallywithin the common knowledge of the skilled artisan (Sambrook et al.,1989).

Having isolated or constructed the nucleotide sequence coding for thenon-bovine pre-prochymosin, prochymosin or chymosin an expression vectoris constructed that comprises the coding sequence and, operably linkedthereto, appropriate expression signals, i.e. sequences to control orregulate the expression, permitting the pre-prochymosin, prochymosin orchymosin to be expressed in a selected host cell. An expression vectorusually includes the components of a typical cloning vector, i.e. anelement that permits autonomous replication of the vector in theselected host organism and one or more phenotypic markers for selectionpurposes. A suitable expression vector may further comprise one or moreexpression signals such as promoter sequences, operators, ribosomebinding sites, translation initiation sites and/or sequences coding forrepressor or activator substances. To permit the secretion of theexpressed polypeptide, a signal sequence may be inserted upstream of thecoding sequence for the pre-prochymosin, prochymosin or chymosin. In thepresent context, the term “expression signal” includes any of the abovecontrol sequences, repressor or activator substances and signalsequences. For expression under the direction of control sequences, thecoding sequence is operably linked to the control sequences in propermanner with respect to expression.

In accordance with the invention, an expression vector carrying thenucleotide sequence coding for pre-prochymosin, prochymosin or chymosincan be any vector that is capable of expressing the coding sequence inthe selected host organism, and the choice of vector type will depend onthe host cell into which it is to be introduced. Thus, the vector may bean autonomously replicating vector, i.e. a vector that exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication in the host cells, e.g. a plasmid, abacteriophage, a minichromosome or an artificial chromosome.Alternatively, the vector may be a vector which, when introduced into ahost cell, is integrated into the host cell genome and replicated withthe chromosome, including a transposable element.

In the vector, the nucleotide sequence coding for the non-bovinepre-prochymosin, prochymosin or chymosin is operably combined with asuitable promoter sequence. The promoter may be any DNA sequence, whichconfers transcriptional activity to the host organism of choice and maybe derived from genes encoding proteins, which are either homologous orheterologous to the host organism. Examples of suitable promoters fordirecting the transcription of the coding sequence of the invention in abacterial host include the promoter of the lac operon of E. coli, thetac promoter, the Streptomyces coelicolor agarase gene dagA promoters,the promoters of the Bacillus licheniformis α-amylase gene (amyL), thepromoters of the Bacillus stearothermophilus maltogenic amylase gene(amyM), the promoters of the Bacillus amyloliquefaciens α-amylase gene(amyQ), the promoters of the Bacillus subtilis xylA and xylB genes andpromoters of lactic acid bacterial origin such as the regulatablepromoters disclosed in WO 94/16086, which is incorporated herein byreference.

For transcription in a fungal species, examples of useful promoters arethose derived from the genes encoding the Pichia pastoris alcoholoxidase, Aspergillus oryzae TAKA amylase, Rhizomucor miehei asparticproteinase, Aspergillus niger neutral-amylase, Aspergillus niger acidstable-amylase, A. niger glucoamylase, A. niger gpdA, A. niger pepA,Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase or Aspergillus nidulansacetamidase, A. nidulans gpdA and a Trichoderma reseei chbl promoter. Asexamples of suitable promoters for expression in a yeast species the Gal1 and Gal 10 promoters of Saccharomyces cerevisiae can be mentioned.When expressed in a bacterial species such as E coli, a suitablepromoter may be selected from a bacteriophage promoter including a T7promoter or a lambda bacteriophage promoter.

The vector comprising the DNA fragment encoding the non-bovinepre-prochymosin, prochymosin or chymosin active polypeptide may alsocomprise a selectable marker, e.g. a gene the product of whichcomplements a defect in the host organism such as a mutation conferringan auxothrophic phenotype, or the marker may be one which confersantibiotic resistance or resistance to heavy metal ions.

In one specific embodiment, the expression vector is derived from pGAMpRas described in Ward et al., 1990 by substituting the coding sequence ofthat vector for bovine prochymosin with a coding sequence for thenon-bovine pre-prochymosin, prochymosin or chymosin. An example of sucha pGAMpR-derived expression vector is pGAMpR-C deposited in anAspergillus niger var. awamori host environment under the accession Nos.CBS 108915 and CBS 108916, respectively.

The person of skill in the art will readily appreciate that any sequencecoding for a non-bovine pre-prochymosin, prochymosin or chymosinconstructable as described above can be modified by substituting,deleting, inserting or adding one or more nucleosides to obtain asequence coding for a non-bovine pre-prochymosin, prochymosin orchymosin comprising the amino acid sequence of the naturally producednon-bovine enzyme or having, relative to the naturally producednon-bovine enzyme, a modified amino acid sequence. Such a modifiedcoding sequence includes a chimeric sequence comprising parts of two ormore coding sequences isolated or derived from non-bovine animal speciesand chimeric coding sequences comprising part of a coding sequence fromone or more non-Bos taurus species and part of a Bos taurus codingsequence.

In a subsequent step of the method a suitable host cell is transformedwith the expression vector. The host cell may be transformed with anautonomously replicating vector or a vector that permits that the codingsequence becomes integrated into the host cell chromosome. Such anintegration is generally considered to be advantageous as the codingsequence is more likely to be stably maintained in the cell. Integrationof the coding sequence into the host chromosome may be carried outaccording to conventional methods such as e.g. by homologous orheterologous recombination or by means of a transposable element.

In accordance with the invention, the host organism may be a cell of ahigher organism such as an animal cell, including a mammal, an avian oran insect cell, or a plant cell. However, in preferred embodiments, thehost organism is a microbial cell, e.g. a bacterial or a fungal cellincluding a yeast cell.

Examples of suitable bacterial host organisms are gram positivebacterial species such as Bacillaceae including Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium and Bacillus thuringiensis, Streptomyces species such asStreptomyces murinus, lactic acid bacterial species includingLactococcus spp. such as Lactococcus lactis, Lactobacillus spp.including Lactobacillus reuteri, Leuconostoc spp. and Streptococcus spp.Alternatively, strains of a gram negative bacterial species such as aspecies belonging to Enterobacteriaceae, including E. coli or toPseudomonadaceae may be selected as the host organism.

A suitable yeast host organism may advantageously be selected from aspecies of Saccharomyces including Saccharomyces cerevisiae or a speciesbelonging to Schizosaccharomyces. Further useful yeast host organismsinclude Pichia spp. such as methylotrophic species hereof, includingPichia pastoris, and Klyuveromyces spp. including Klyuveromyces lactis.

Suitable host organisms among filamentous fungi include species ofAcremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophtora,Neurospora, Penicillium, Thielavia, Tolypocladium or Trichoderma, suchas e.g. Aspergillus aculeatus, Aspergillus awamori, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus oryzae, Aspergillusnidulans or Aspergillus niger, including Aspergillus niger var. awamori,Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichiodes, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola insolens,Humicola langinosa, Mucor miehei, Myceliophtora thermophila, Neurosporacrassa, Penicillium chrysogenum, Penicillium camenbertii, Penicilliumpurpurogenum, Rhizomucor miehei, Thielavia terestris, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesii or Trochoderma viride.

Examples of Aspergillus niger var. awamori strains transformed with avector expressing non-bovine pre-prochymosin, prochymosin or chymosininclude the strains deposited under the accession Nos. 108915 and108916.

Some of the above useful host organisms, such as fungal species or grampositive bacterial species, may be transformed by a process whichinvolves protoplast formation and transformation of the protoplastsfollowed by regeneration of the cell wall in a manner known per se.

In subsequent steps the thus transformed host cell is cultivated underconditions where the coding sequence is expressed, and thepre-prochymosin, prochymosin or chymosin is harvested. The medium usedto cultivate the transformed host cells may be any conventional mediumsuitable for growing the host cells in question and obtaining expressionof the polypeptide. Suitable media are available from commercialsuppliers or can be prepared according to published recipes.

The resulting non-bovine pre-prochymosin, prochymosin or chymosin istypically recovered or harvested from the cultivation medium byconventional procedures including separating the host cells from themedium by centrifugation or filtration, if necessary, after disruptionof the cells, followed by precipitating the proteinaceous components ofthe supernatant or filtrate e.g. by adding a salt such as ammoniumsulphate, followed by a purification step. Alternatively, the cell-freecultivation medium may also, optionally after concentrating or dilutingit or addition of conventional additives, be used directly as acoagulant product for cheese manufacturing.

It will be appreciated that the non-bovine pre-prochymosin, prochymosinor chymosin as isolated can be subjected to sequence modifications bydeleting, substituting, modifying or adding one or more amino acids aslong as the resulting modified molecule retains at least part of themilk clotting activity of the non-bovine pre-prochymosin, prochymosin orchymosin as isolated. Such modifications can readily be performed by theperson of skill in the art using methods for protein modifications thatare commonly known in the art.

In accordance with the invention, the nucleotide sequence coding fornon-Bos taurus pre-prochymosin, prochymosin or chymosin is isolated orderived from a mammalian species selected from the group consisting of aruminant species, a Camelidae species, a porcine species, an Equidaespecies and a primate species. A ruminant species source animal may beselected from camel species, deer species, buffalo species, antelopespecies, giraffe species, ovine species and caprine species. Aparticularly interesting source animal is Camelus dromedarius.

It has been found that expression and secretion of a heterologous geneproduct can be enhanced by expressing the gene product in the form of afusion protein. In this context, the term “fusion protein” denotes achimeric protein comprising pre-prochymosin, prochymosin or chymosin orat least a milk coagulation active part hereof and, as the fusionpartner, at least one amino acid of a different polypeptide.Accordingly, in one embodiment of the invention, the above method is onewherein the nucleic acid sequence codes for a fusion protein comprisingpre-prochymosin, prochymosin or chymosin. More specifically, the fusionpartner may be glucoamylase or a fragment thereof. In one embodiment thepre-prochymosin, prochymosin or chymosin, or a fusion protein thereof,is secreted over the host cell membrane.

One major objective of the present invention is to provide a method ofproducing a non-bovine pre-prochymosin, prochymosin or chymosinrecombinantly at a high yield. During the experimentation leading to theinvention it was a highly unexpected finding that a non-bovinepre-prochymosin, prochymosin or chymosin, when expressed in the samehost cell and under essentially identical conditions, is expressed atactivity yields which are significantly higher than are the obtainedactivity yields of bovine chymosin.

In accordance herewith, the above method of the invention is preferablya method wherein the yield of non-bovine pre-prochymosin, prochymosin orchymosin milk clotting activity is at least 10%, 25%, 50%, 100% or 200%higher than the yield of bovine pre-prochymosin, prochymosin or chymosinmilk clotting activity obtained when using, under identical productionconditions, the same expression vector, but with a coding sequence forbovine pre-prochymosin, prochymosin or chymosin in place of the sequencecoding for the non-bovine pre-prochymosin, prochymosin or chymosin.

It is generally known that polypeptides expressed by eukaryotic hostorganisms may be glycosylated when expressed, the degree ofglycosylation depending on the type of polypeptide and host organism. Ithas been found previously that the milk clotting activity of asparticproteases of microbial origin that are glycosylated upon expression maybe enhanced by subjecting the proteases to a deglycosylating treatmentto at least partially remove the sugar moieties attached to theproteases. Such a deglycosylation treatment may e.g. comprise treatingthe glycosylated protease with an enzyme having a deglycosylatingactivity including as examples PNGase and endo-β-N-acetylglucosaminidase(EC 3.2.1.96) (Endo-H). Alternatively, the deglycosylation may beobtained by subjecting the glycosylated protease to a chemicaltreatment, such as treatment with periodate.

Accordingly, in a specific embodiment, the above method comprises, as afurther step, that the harvested pre-prochymosin, prochymosin orchymosin is subjected to a deglycosylation treatment.

It is also contemplated that deglycosylation of an expressedpre-prochymosin, prochymosin or chymosin can be obtained in a moredirect manner by providing a host cell that in addition to thepre-prochymosin, prochymosin or chymosin expresses a deglycosylatingenzyme such as Endo-H whereby the initially glycosylatedpre-prochymosin, prochymosin or chymosin is deglycosylatedintracellularly or following secretion. Accordingly, in a anotherembodiment the host cell is a cell further expressing a deglycosylatingenzyme capable of deglycosylating co-expressed pre-prochymosin,prochymosin or chymosin.

In another aspect, the invention provides a DNA construct capable ofexpressing non-bovine pre-prochymosin, prochymosin or chymosin. Thisconstruct comprises an expression vector comprising a nucleic acidsequence comprising a gene coding for the pre-prochymosin, prochymosinor chymosin and, operably linked thereto, appropriate expression signalsas defined above, permitting the pre-prochymosin, prochymosin orchymosin to be expressed in a host cell. Accordingly, such a constructincludes a construct that comprises a sequence coding for a signalpeptide for the pre-prochymosin, prochymosin or chymosin and/or anexpression signal that is a promoter not natively associated with thecoding sequence.

The coding sequence of the DNA construct of the invention can be derivedfrom any of the above non-bovine, i.e. non-Bos taurus, animal speciesincluding Camelus dromedarius. In useful embodiments, the DNA constructcomprises a nucleic acid sequence that codes for a fusion protein asalso defined above, comprising the pre-prochymosin, prochymosin orchymosin or a fragment hereof having milk clotting activity. In afurther embodiment, the the fusion protein comprises glucoamylase or afragment thereof. The expression vector of the DNA construct may be anyof the expression vectors mentioned above including pGAMpR-derivedvectors such as the pGAMpR-C vector as described in the below examples.Additionally, the DNA construct according to the invention may furthercomprise a sequence coding for a deglycosylating enzyme such as endoH.

The sequence of the DNA construct according to the invention that codesfor a non-bovine pre-prochymosin, prochymosin or chymosin may be anaturally occurring coding sequence. However, as it will be appreciatedby the person of skill in the art, the coding sequence may also be onethat is derived from such a naturally occurring coding sequence by oneor more silent nucleotide substitution(s), the term “silent” implyingthat the codon in which the substitution(s) occur codes for the sameamino acid as the corresponding codon in the naturally occurring codingsequence.

In a further aspect, the invention provides a host cell transformed witha DNA construct as described above. The host cell is selected from anyof the above organisms, i.e. bacterial cells, fungal cells includingAspergillus niger var. awamori such as the strains deposited as CBS108915 and CBS 108916., yeast cells, mammalian cells, insect cells andplant cells.

In a still further aspect a milk clotting composition is providedcomprising a non-bovine prochymosin or chymosin as defined herein andproduced by the above method including such a prochymosin or chymosinthat is in a substantially deglycosylated form. Such a composition may,in addition to the active milk clotting enzyme, comprise additives thatare conventionally used in rennets of animal origin such as e.g. NaCl.In preferred embodiments, the composition comprises pre-prochymosin,prochymosin or chymosin derived from the group consisting of a Camelidaespecies, a buffalo species, an ovine species or a caprine species.

The recombinant non-bovine pre-prochymosin, prochymosin or chymosin asprovided herein is useful as a milk coagulant product. Accordingly, animportant objective of the invention is to provide a method ofmanufacturing cheese, comprising adding a milk clotting effective amountof the above composition to milk and carrying out appropriate furthercheese manufacturing steps. The pre-prochymosin, prochymosin or chymosinof the invention is suitable for cheese manufacturing processes whereinthe milk is selected from cow's milk, camel milk, buffalo milk, goat'smilk and sheep's milk.

An aspartic protease such as chymosin that is suitable for cheesemanufacturing should have a high specific milk clotting activity (C) anda low general, i. e. non-specific, proteolytic activity (P) with regardto milk proteins. Accordingly, the C/P ratio should preferably be ashigh as possible, as a relatively high P-value, during the cheesemanufacturing pro??cess and during maturation of the cheese will lead tothe formation of low molecular peptides and free amino acids, which inturn may confer to the finished cheese an undesirable bitter taste andalso result in a loss of cheese yield. As used herein, the term “C/Pratio” is defined by the methods for determining a C-value and aP-value, respectively as described in the below examples.

As shown in the below Examples, the use of a recombinantly producednon-bovine prochymosin or chymosin in cheese manufacturing results in ahigher yield of cheese than the yield obtained with the same amount ofmilk clotting activity of bovine prochymosin or chymosin. Accordingly,in one embodiment the invention provides a cheese manufacturing methodwherein the yield of cheese obtained is higher than the yield obtainedunder identical manufacturing conditions using the same amount of bovineprochymosin or chymosin.

It is demonstrated in the below examples, that the non-bovinepre-prochymosin, prochymosin or chymosin has a higher C/P ratio,relative to the conventionally used bovine chymosin. Accordingly, theinvention pertains in yet another aspect to a method of manufacturingcheese, comprising adding a milk clotting effective amount of anon-bovine prochymosin or chymosin to the milk including bovine milk andcarrying out appropriate further cheese manufacturing steps, thenon-bovine prochymosin or chymosin having in said milk a C/P ratio inthe range of 2-20, preferably a C/P ratio of at least 3, such as atleast 5 or even at least 10.

In one specific embodiment such a pre-prochymosin, prochymosin orchymosin is derived from Camelus dromedarius.

It is a further objective of the invention to provide a milk clottingcomposition comprising a milk clotting bovine enzyme selected fromprochymosin, chymosin and pepsin and a non-bovine milk clotting enzymeselected from prochymosin, chymosin and pepsin including such acomposition where the milk clotting activity ratio between the bovineand the non-bovine milk clotting enzyme is in the range of 1:99 to 99:1,including a composition where at least 2% of the milk clotting activityis from the non-bovine enzyme such as at least 5%, 10%, 20%, 50%, 75, 90or 98% of the activity. In one preferred embodiment, the non-bovineenzyme in such a mixed composition is derived from Camelus dromedarius.

There is also provided a method of manufacturing cheese from milkincluding cow's milk, camel's milk, buffalo milk, goat's milk, sheep'smilk and a mixture of any such milk types, comprising adding a milkclotting effective amount of the above composition and carrying outappropriate further cheese manufacturing steps.

The invention will now be described in further details in the following,non-limiting examples and the drawings where:

FIG. 1 is a map of plasmid pSK-SbXb-mut containing a glucoamylaseA-chymosin B expression cassette. A silent mutation results in a uniquePmlI site;

FIG. 2 is a map of plasmid pGAMpR-pmI which is identical to plasmidpGAMpR except for a silent mutation resulting in a unique PmlI site;

FIG. 3 is a map of plasmid pGAMpR-C, a camel chymosin expressionconstruct;

FIG. 4 shows the milk clotting activity in arbitrary units insupernatants of plasmid pGAMpR-C transformed Aspergillus niger var.awamori cultivated in shake flasks in CSL medium for 24-48 hrs at 37° C.and 200 rpm;

FIG. 5 shows the production of camel chymosin activity in pilot scalefermentation of pGAMpR-C transformed Aspergillus niger var. awamoristrain #21 and #28 as compared to production of bovine chymosin activityusing an Aspergillus niger var. awamori strain transformed with pGAMpR;

FIG. 6 illustrates the progress of whole casein digestion over timeusing recombinantly produced camel chymosin (▪------▪) and bovinechymosin (♦------♦), respectively;

FIG. 7 illustrates the general, non-specific proteolytic activity asabsorbancy of 3% TCA-precipitated supernatant of 100 nM recombinantlyproduced camel chymosin (▪------▪) and bovine chymosin (♦------♦),respectively in 33 nM MES, pH 5.80 using 0.5% N,N-demethylated bovinecasein as the substrate and incubating at 32° C. for 30-180 min. atdifferent pH;

FIG. 8 illustrates the general, non-specific proteolytic activity asabsorbancy of 3% TCA-precipitated supernatant of 100 nM recombinantlyproduced camel chymosin (▪------▪) and bovine chymosin (♦------♦),respectively in 33 nM MES, pH 5.80 using 0.5% N,N-demethylated bovinecasein as the substrate and incubating at 32° C. for 30-180 min. atdifferent Ca²⁺ concentrations;

FIG. 9 illustrates the general, non-specific proteolytic activity asabsorbancy of 3% TCA-precipitated supernatant of 100 nM recombinantlyproduced camel chymosin (▪------▪) and bovine chymosin (♦------♦),respectively in 33 nM MES, pH 5.80 using 0.5% N,N-demethylated bovinecasein as the substrate and incubating at 32° C. for 30-180 min. atdifferent temperatures; and

FIG. 10 shows peptide and protein concentration in 50 μl of the solublephase of a coagulum of reconstituted bovine skimmed milk coagulated for5, 60 and 1320 min., respectively with 65 mlMCU ml⁻¹ recombinantlyproduced camel chymosin (▪------▪) and bovine chymosin (♦------♦),respectively, measured as absorbancy at 280 nm, and diluted in 950 μl of8M guanidine-HCl.

EXAMPLE 1 Construction of a Vector for the Expression of Camel Chymosin

Unless indicated otherwise, recombinant DNA techniques were according toSambrook et al., 1989.

1.1 Cloning of Camelus dromedarius Chymosin Gene

A DNA sequence containing a camel prochymosin (cd-prochymosin) codingsequence and adjacent 5′ and 3′ sequences of the pGAMpR vector (Ward etal., 1990) was amplified by PCR. The pGAMpR vector comprises, as aselection marker, the pyr4 gene of Neurospora crassa, which is capableof complementing a pyrG mutation in a recipient strain. mRNA wasisolated from mucosal tissue of a 3 year old camel using a direct mRNAKit (Quiagen, D-40724 Hilden, Germany). Based on this isolated mRNA, acDNA template for PCR was generated by reverse transcription. For PCRamplification the following pair of primers were used:

-   cd-prochymosin forward:    -   PmlI-   5′-cacgtggcggAGTGGGATCACCAGGATCCCTCTG-3′ (SEQ ID NO:1)-   cd-prochymosin reverse:    -   XbaI-   5′-tctagaggaTCAGATGGCCTTGGCCAGCCCCACG-3′ (SEQ ID NO:2)

The PCR product was ligated into a pCR-script vector (Stratagene, LaJolla, Calif.) according to the manufacturer's recommendations.

1.2 Construction of cd-prochymosin Expression Vector, pGAMpR-C

For construction of pGAMpR-C, a SpeI-XbaI fragment containing a fusionbetween the Aspergillus niger glucoamylase and the Bos taurusprochymosin coding sequences was isolated from pGAMpR. This fragment wascloned into pBluescript-SK II+, resulting in vector pSK-SpXb.

A silent mutation was introduced into pSK-SpXb by oligonucleotide-basedmutagenesis in the 3′-region of the glucoamylase to create a unique sitefor PmlI, resulting in plasmid pSK-SpXb-mut (FIG. 1). The following pairof oligonucleotide primers was used (bases introducing the mutation inthe vector are shown in capitals):

5′-gcgacggtgactgacacGtggcgggcagaaata (SEQ ID NO: 3) ac-3′ (PmlI mutationforward) 5′-gttatttctgcccgccaCgtgtcagtcaccgtc (SEQ ID NO: 4) gc-3′ (PmlImutation reverse)

The SpeI-XbaI fragment from pSK-SpXb-mut was used to replace thecorresponding fragment in pGAMpR, resulting in pGAMpR-pmI (FIG. 2).pGAMpR-pmI was digested with PmlI and XbaI and the camel cDNA clone,digested with the same restriction enzymes, was inserted, resulting inpGAMpR-C (FIG. 3). The sequence of the glucoamylase-camel prochymosinfusion in pGAMpR-C was confirmed on both strands.

Accordingly, the Aspergillus expression vector plasmid pGAMpR-C isidentical to pGAMpR, the expression plasmid used for expression ofbovine chymosin, except that the prochymosin coding sequence is thatisolated from Camelus dromedarius. When expressed and secreted, thecamel prochymosin is converted into chymosin by autocatalytical cleavageof the pro-sequence.

EXAMPLE 2 Transformation of Aspergillus niger var. awamori with pGAMpR-C

For these transformation experiments, a derivative of Aspergillus nigervar. awamori, strain dgr246pyrG (Ward et al., 1993) was used asrecipient. This strain is a derivative of Aspergillus niger var. awamoristrain GCI-HF1-2dgr246 having a pyrG mutation, rendering the strainincapable of growing in the absence of uridine, and which comprisesseveral copies of the pGAMpR plasmid. The derivative strain, dgr246pyrGused as recipient is derived by curing the pyrG mutant parent strain forall copies of pGAMpR.

The dgr246pyrG strain has been deposited under the Budapest Treaty withthe Centraal-bureau voor Schimmelcultures (CBS), Oosterstraat 1, P.O.Box 273, 3740 AG Baarn, The Netherlands, on 13 Jun. 2000 under theaccession No. CBS 108914.

An optimised protocol as developed by Chr. Hansen A/S was applied fortransformation of the “cured” Aspergillus strain. This protocolcomprises the steps of providing a liquid culture medium, propagation offungal biomass, generation of protoplasts and transformation includingregeneration of protoplasts and selection of transformants.

2.1 Propagation of Fungal Biomass

50 ml of CSL medium [per litre: corn steep liquor, 100 g; NaH₂PO₄.2H₂O,1 g; MgSO₄, 0.5 g; Mazu antifoaming agent, 2 g, maltose, 100 g, glucose,10 g, fructose, 50 g, water 736.5 g] is added to a sterile 250 ml flask,0.5 ml penicillin/streptomycin supplement (Gibco-BRL #15140-114)] isadded and the medium inoculated with 10⁶ spores per ml. The inoculatedmedium is cultivated overnight at 34-37° C. at 200-250 rpm to obtain adense suspension of mycelium. 10 ml of this pre-culture is transferredto 100 ml complete Aspergillus medium in a 500 ml flask without baffles,incubation overnight at 34-37° C. at 200-250 rpm to obtain a mycelialbiomass.

2.2 Generation of Protoplasts

Mycelium as obtained in the above step is filtered over sterilemyracloth, washed with sterile water and subsequently with 1700 mOsmolNaCl/CaCl₂ (0.27 M CaCl₂.2 H₂O, 39.7 g/l; 0.58 M NaCl, 33.9 g/l), gentlysqueezed dry and transferred to a Falcon tube to determine the weightand left to stand on ice.

20 ml 1700 mOsmol NaCl/CaCl₂ per g mycelium is added to resuspend themycelium followed by adding 50 mg Sigma L-1412 Trichoderma harzianumLytic Enzyme per g mycelium dissolved in a small volume of 1700 mOsmolNaCl/CaCl₂, incubation in an Erlenmeyer flask at 100 rpm, 37° C. forabout 4 hrs during which period the mycelium is repeatedly resuspendedevery 30 minutes.

When good protoplasting is obtained, i.e. many free protoplasts occurand with hardly any intact mycelium left, the mixture is filtered onice, using Mesh sheet or myracloth and an equal volume of ice coldSTC1700 (1.2 M sorbitol, 218 g/l; 35 mM NaCl, 2.04 g/l; 10 mM Tris.HClpH 7.5 and 50 mM CaCl₂.2H₂O, 7.35 g/l) is added. The number ofprotoplasts is counted using a glass Bürger-Türk chamber. The protoplastsuspension is spun using a bench top centrifuge at 2,000 rpm at 4° C.The resulting pellet is resuspended very gently in 20 ml ice coldSTC1700. This washing procedure is repeated twice and the final pelletis resuspended in ice cold STC1700 to a final concentration of about1×10⁸ protoplasts per ml followed by adjustment to 1×10⁸ protoplasts perml.

2.3 Transformation

200 μl (2×10⁷ protoplasts), 2 μl of 0.5 M ATA (0.5 M aurine carboxylicacid (Sigma) in 20% ethanol) and DNA (comprising a marker) up till 15μl, typically 5-10 μg of DNA, is mixed in a 12 ml test tube. As controla corresponding mixture, but without DNA is used. The transformationmixtures are incubated on ice for 25 min. followed by adding a firstdrop of 250 μl PTC (60% PEG 4000; 10 mM Tris.HCl pH 7.5; 50 mM CaCl₂) bytipping the tube a couple of times without letting the mixture touch thelid and a second drop of 250 μl, mixing and adding 850 μl followed bymixing. Each tube is incubated at room temperature exactly 20 min.followed by filling the tubes with ice cold STC1700 and mixing byreverting the tubes. The mixture is centrifuged for 8-10 min. using abench top centrifuge at 2000 rpm at 4° C. The resulting pellet isdissolved gently in about 400-800 μl STC1700.

2.4 Regeneration and Selection of Transformants

The transformation mixture is spread onto solid selective regenerationmedium plates containing per l medium: agar, 15; sorbitol, 218 g; AspAsalts 50× (per litre: 300 g NaNO₃, 26 g KCl, 76 g KH₂PO₄, 18 ml 10 MKOH, pH about 6.5); glucose 50%, 20 ml; Gibco-BRL #15140-114 Pen-Strep,10 ml; MgSO₄, 2 ml; trace elements (2.2 g ZnSO₄, 1.1 g H₃BO₃, 0.5 gMnCl₂.7H₂O, 0.5 g FeSO₄.7H₂O, 0.17 g CoCl₂.6H₂O, 0.16 CuSO₄.5H₂O, 0.15NaMoO₄.2H₂O, 5 g EDTA, water to 100 ml, pH 6.5), 1 ml. The plates areincubated at 37° C. for 5-10 days and transformants selected.

About 80 transformants were obtained and spores of these transformantswere obtained.

EXAMPLE 3 Production of Camel Chymosin Using Recombinant Aspergillusniger var. awamori

3.1 Selection of Transformants Producing High Amounts of Chymosin

To select the transformants that produced the most chymosin, a smallscale (20 ml) shake flask experiments with 45 transformants was carriedout. 20 ml of CSL medium (see above) was inoculated with 1×10⁸ spores ofeach transformant, incubation 24-48 hrs at 37° C. and 200 rpm. 2 ml ofthese precultures was used for inoculation of 20 ml medium followed byincubation for 10 days at 37° C., 200 rpm. After incubation the cultureswere centrifuged at 14,000 rpm using an Eppendorf centrifuge and theclear supernatants were collected and stored at −20° C. untildetermination of chymosin activity. As controls, both the recipientstrain and an Aspergillus niger var. awamori production strain forbovine chymosin, dgr246chlor25 (Dunn-Coleman et al., 1991) containingthe pGAMpR (spores of this strain used as inoculum material is referredto herein as PIM2075) were included.

The results of these experiments are summarised in FIG. 4. It appearsthat 5 of the tested strains produced in excess of 40 arbitrary unitsper ml supernatant. Among the tested transformants, the best producerswere strains #21 and #28.

A sample of the strains #21 and #28 were deposited under the BudapestTreaty with the Centraalbureau voor Schimmelcultures (CBS), Oosterstraat1, P.O. Box 273, 3740 AG Baarn, The Netherlands, on 13 Jun. 2000 underthe accession Nos. 108915 and 108916, respectively.

A colony PCR experiment was carried out to verify that the chymosinproduced by the transformants in fact was camel chymosin. Mycelium ofthe transformants was analysed using two primer sets, one specific forbovine chymosin and one specific for camel chymosin. It was confirmedthat all transformants only contained the Camelus dromedarius gene inthat no bands were observed in any of the transformants using the bovineprimer set, but bands were generated in all of the transformants whenthe camel chymosin primer set was used. Both the cured recipient strainand the bovine chymosin production strain were tested similarly. Nobands could be observed with either primer set when the cured strain wastested whereas the control production strain yielded PCR products onlywith the bovine chymosin primer set.

3.2 Pilot Scale Production of Camel Chymosin

The two best produces from the above small scale screening procedurewere tested further for their chymosin production capabilities in 19 lBioengineering NLF22 fermentors. As a control, an Aspergillus niger var.awamori strain transformed with pGAMpR, referred to as PIM2075 wastested similarly.

The basic medium used in this experiment had the following compositionper litre: Dan-pro™ soya, 44.68 g; KH₂PO₄, 1.06 g; Mazu DF204Kantifoaming agent, 1.00 g; MgSO₄.7H₂O, 2.07 g; NaH₂PO₄.2H₂O, 1.20 g;(NH₃)₂PO₄, 17.29 g; H₂SO₄ 38%, 0.80 ml, water to 1 litre. A 35% aqueoussolution of maltose was used as the carbon source and to maintain the pHat the pre-set value, a 25% NH₃ solution was used. The fermentationparameters were the following: pH: 5.5; temperature: 35° C.; agitation:600 rpm; air supply: 12 l per min.; overpressure: 0.5 bar.

During 164 hrs of fermentation, the concentration of camel and bovinechymosin activity, respectively was determined at intervals in thefermentation broths. The results are summarised in FIG. 5. As itappears, the yield of camel chymosin activity from both transformantstrains was, during the entire fermentation period, significantly higherthan that of bovine chymosin. At the end of the fermentations, thechymosin milk clotting activity yield of recombinant strains #21 and #28was 361 and 343 arbitrary units, respectively, whereas the yield ofbovine chymosin activity produced by PIM2075 was 215 arbitrary units,i.e. the recombinant strains expressing Camelus dromedarius chymosinproduced about 70% more chymosin activity than the bovine chymosinproducing strain did under identical production conditions.

EXAMPLE 4 Non-specific Proteolytic Activity of Recombinant Camelusdromedarius Chymosin

In this experiment, the general (non-specific) proteolytic activity(P-value) of recombinantly produced Camelus dromedarius chymosin (Cdchymosin) as obtained in Example 3 on bovine whole casein was studied.For comparison, a recombinantly produced bovine chymosin, ChyMax™ (Btchymosin) was included. The P-value was tested over time and the effecton the proteolytic activity of Ca²⁺ and pH, respectively, was studied.

4.1 Assay

Unless otherwise stated, the activity reactions were done under thefollowing conditions: 0.5% N,N-dimethylated bovine casein (Sigma C9801),100 nM chymosin in 33 nM MES, pH 5.80 at 32° C. for 30 min. to 180 min.The absorbency of 3% TCA supernatants was measured at 280 nm. Theconditions for the activity reactions were selected so as to includethose of a conventional cheese manufacturing process in respect ofsubstrate to be analysed, concentrations of substrate and enzyme,temperature (30-35° C.), pH (about 6.6), Ca²⁺ concentration (0-2 nM) andreaction time.

4.2 Results

Over a time period of 180 min., the P-value of the Bt chymosin increasedprogressively to an A₂₈₀ of about 0.5 whereas, during the reactionperiod the Cd chymosin showed a much lower non-specific proteolyticactivity, ending at an A₂₈₀ of about 0.1 (FIG. 6).

The effects of pH in the range of 5.4-6.8, Ca²⁺ concentration in therange of 0-8 mM and the temperature in the range of 30-65° C., on thenon-specific proteolytic activity of Cd chymosin and Bt chymosin,respectively are summarised in FIGS. 7-9. As it appears, thenon-specific proteolytic activity of the Camelus dromedarius chymosinwas generally significantly lower that that of the bovine chymosin underall test conditions used.

A higher non-specific proteolytic activity was observed when the pH ofthe assay was lowered (FIG. 7). The relation of the P-values betweencamel and bovine chymosin remained substantially constant over the pHrange studied.

EXAMPLE 5 The Milk Clotting Activity of Recombinant Camelus dromedariusChymosin

In this experiment, the milk clotting activity (C-value) of therecombinant Cd chymosin of the invention was studied at 32° C. using assubstrate 10% (w/v) low heat spray-dried bovine skimmed milk (Hochdorf).The concentration of Cd chymosin used was 3.1 nM. For comparison, theC-value of recombinant Bt chymosin (ChyMax™) at a concentration of 5.4nM (same milk clotting activity in IMCUs as the Cd chymosin) wasdetermined under the same conditions. The milk clotting activity andfinal curd strength were determined. In addition, the effect of changesin pH and Ca²⁺ concentration on the milk clotting activity of bothenzymes was determined. The results of these experiments can besummarised as follows:

The milk clotting activity of the camel chymosin is less affected bychanges in pH and Ca²⁺ concentration. The average clotting activity (C)per mole of camel chymosin was about 170% of the activity per mole ofthe bovine chymosin. Under typical cheese manufacturing conditions asdescribed above, the camel chymosin C-value is about 180% of thecorresponding value for bovine chymosin. The final milk curd strengthwas essentially the same for both enzymes, indicating similar clottingand synthesis reactions.

Based on the results in this Example and those found in Example 4, theC/P ratio for camel chymosin can be estimated, relative to that forbovine chymosin. These data are summarised in the below table 5.1 whichalso shows corresponding data for porcine chymosin, bovine pepsin B andtwo microbial aspartic proteases:

TABLE 5.1 Milk clotting activity, non-specific proteolytic activity andC/P value for porcine chymosin, bovine pepsin B and two microbialaspartic proteases: Milk clotting Non-specific activity proteolytic (%of activity (% bovine chymosin) of bovine chymosin) C/P value Bovinechymosin 100 100 1.00 Camel chymosin 170 25 7.00 Porcine chymosin 25 122.10 M. pusillus protease 33 147 0.22 M. miehei protease 19 149 0.13Bovine pepsin A 124 2731 0.05

These data shows that among the listed aspartic proteases, recombinantcamel chymosin has the highest milk clotting activity and the secondlowest non-specific proteolytic activity on bovine casein, resulting inby far the highest C/P value among these proteases. In particular, it issignificant that in respect of these parameters, camel chymosinoutperforms the commercial recombinant bovine chymosin significantly.

EXAMPLE 6 Determination of the Amount of Enzymatic Digests and WheyProtein in the Soluble Phase of Milk Coagulum Using Recombinant Camelusdromedarius (Cd) Chymosin or ChyMax™ (Bt Chymosin)

10% (w/v) spray-dried bovine skimmed milk was dissolved in 50 mM MES, 1mM CaCl₂, pH 5.6. The milk was coagulated with 65 mlMCU ml⁻¹ of therespective recombinant chymosin preparations, i.e. recombinant Camelusdromedarius (Cd) chymosin or ChyMax™ (Bt chymosin) for 5, 60, 180 and1320 min., respectively. The formed coagula were centrifuged at 20,000×gat 4° C. for 5 min. Fifty μl of the supernatant containing breakdownproducts of enzymatic digests and whey proteins was diluted in 950 μl of8M guanidine-HCl, pH 6.5 and the absorbancy at 280 nm was measured.

The results are summarised in FIG. 10. As it appears, the amount ofpeptides and protein was highest in supernatants derived fromcoagulation with the bovine chymosin, indicating that the camel chymosinunder milk clotting conditions has less non-specific proteolyticactivity than bovine chymosin.

EXAMPLE 7 Coagulation of Raw Cow's Milk and Camel Milk UsingRecombinantly Produced Recombinant Camelus dromedarius (Cd) Chymosin orChyMax™ (Bt Chymosin)

Raw milk was stored for two days at 4° C. Milk samples (10 per analysis)were coagulated with 65 mlMCU ml⁻¹. The rennet coagulation time (r[min])and the curd strength of the coagula (A₆₀[mm]) were determined. Curdstrength was determined using a Formagraph device (Foss Electric,Hillerød, Denmark). The results are summarised in Table 7.1.

Dilutions of camel and bovine chymosin adjusted to the same clottingactivity on bovine spray-dried skimmed milk in 1 mM CaCl₂ showedactivity on raw bovine milk which was similarly higher (shorter rennetcoagulation time) for both enzymes, relative to the activity on thespray-dried bovine milk. The strength of the final curd of bovine milkwas slightly higher when using the camel chymosin. Bovine chymosinhardly had any renneting activity on raw camel milk, whereas camelchymosin showed a high activity resulting in a curd of medium curdstrength.

TABLE 7.1 Rennet coagulation time and curd strength using bovine orcamel chymosin Enzyme Milk r[min] A₆₀[mm] Bt chymosin Bovine raw milk12.34 48.94 Cd chymosin Bovine raw milk 12.20 50.56 Cd chymosin Bovineskimmed milk 22.07 37.96 Bt chymosin Camel raw milk 59.45 0.40 Cdchymosin Camel raw milk 12.66 18.24

EXAMPLE 8 Examination of the Proteolytic Activity of Recombinant Camelusdromedarius (Cd) Chymosin and ChyMax™ (Bt Chymosin)

Two synthetic peptidases, which correspond to the chymosin sensitiveregions of camel- and bovine κ-casein (CN), were proteolytically cleavedwith bovine and camel chymosin, respectively. K_(M) and k_(cat) weredetermined at pH 5.6. Additionally, temperature and pH optima of thesereactions were measured. The two following synthetic peptides were usedin this study:

Synthetic Peptide Corresponding to Bovine κ-CN

-   NH₂-His-Pro-His-Pro-His-Leu-Ser-(p-NO₂)Phe∞Met-Ala-Ile-COOH (SEQ ID    NO:5)    Synthetic Peptide Corresponding to Camel κ-CN-   NH₂-Arg-Pro-Arg-Pro-Arg-Pro-Ser-(p-NO₂)Phe∞Ile-Ala-Ile-COOH (SEQ ID    NO:6)-   ∞ fissile bond

The measurement of samples was repeated 3 to 6 times, theMichaelis-Menten and the turnover values were determined fromLineweaver-Burke plots with weighted linear regression of data.

The results are summarised in Table 8.1

TABLE 8.1 Summary of results Bt Chymosin Cd Chymosin K_(M) for Bt κ-CN0.165 + 0.015/−0.014 mM 0.077 + 0.019/−0.015 mM k_(cat) for Bt κ-CN 44.3 + 1.3/−1.2 s⁻²  11.7 + 1.5/−1.2 s⁻¹ K_(M) for Cd κ-CN 0.134 +0.022/−0.021 mM 0.056 + 0.035/−0.021 mM k_(cat) for Cd κ-CN  4.3 +0.2/−0.1 s⁻²  5.1 + 1.7/−1.0 s⁻¹ Temp. optimum (Bt κ-CN ~42° C. ~42° C.Temp. optimum (Cd κ-CN) ~58° C. ~47° C. pH optimum (Bt κ-CN) ~4.9 ~5.1

Marked differences were found when the enzymes were examined forproteolytic activity towards two synthetic peptides representing part ofthe chymosin sensitive regions of bovine and camel κ-casein. Thesubstrate binding of camel chymosin was found to be about double as high(half K_(M) value) as the substrate binding of bovine chymosin and theturn-over rate of camel chymosin against the bovine κ-CN peptide wasabout four times lower than the turnover rate of bovine chymosin.

These findings may explain the higher C/P ratio found for camel chymosinin Example 5. Since the fissile bond of κ-CN represents only a smallfraction of the fissile sites in milk proteins, a higher specificbinding of the target molecule, and a low non-specific proteolyticactivity, effecting self-inhibition of the enzyme and subsequentactivation by the target molecule, will lead to a high C/P ratio.Furthermore, similar temperature and pH optima were found forproteolysis of the Bt κ-CN peptide, and the temperature optima for theCd κ-CN peptide were found to be markedly higher, mainly the one ofbovine chymosin.

EXAMPLE 9 Determination of the Cheese Yield Using ChyMax™ (FPC) andCamel Chymosin (FPCC)

9.1 Introduction

The effect of milk clotting enzymes on cheese yield is a characteristicof great commercial importance. Careful measurement of the level of drymatter in whey is used as a method of comparing the effect of enzymes oncheese yield. In this study the cheese yield obtained by using thecommercial product ChyMax™ (FPC) and the fermented produced camelchymosin (FPCC), both of Chr. Hansen A/S origin, were compared.

9.2 Materials and Methods

9.2.1 Milk Clotting Enzymes

The milk clotting enzymes used were FPC (batch no. 2114475, 198 IMCU/ml)and fermented produced camel chymosin, FPCC, (batch no. SR 30.05.00, 234IMCU/ml). The amounts used were such that a cutting time of 30 minuteswere obtained. Dosages were kept constant throughout the study.Variation in the coagulability of the milk were compensated by varyingcutting times.

9.2.2 Cheese Making in Beakers

Whole pasteurised non-homogenised milk from Arla Foods, Slagelse,Denmark, was used. The cheese making procedure is summarised in Table9.1. 4000 g of milk was added to a 5-litre beaker. GluconoDeltaLactone(GDL) from “DAN BOUQUET” was added in an amount of 3,200 g. 1,600 g ofCaCl₂ was added. Cutting took place after about 30 min. Curd and wheywere transferred to cheesecloth after healing and stirring for 30 min,and left to drain overnight. Both milk clotting enzymes were used ineach of the 16 trials.

9.2.3 Sampling and Analysis

Well mixed total whey was filtered through a layer of gauze to removecheese dust. Dry matter was determined by drying about 25 g of whey onlapis pumices p.a. for 4 hours at a temperature of 110° C. The analyseswere done in duplicate.

9.2.4 Statistical Analysis

A paired students t test was used on n=16 differences in total drymatter of the whey, usingt=x _(mean) /s ₂ /n)_(1/2)as an estimate,t ₁₆(90%)=1.34, t ₁₆(95%)=1.75, t ₁₆(99.5%)=2.929.3 Results and Discussion

Table 9.1 summaries some parameters for the cheese making procedure.Recovery of milk as cheese and whey was over 99%. Clotting activity of140 IMCU FPC and 140 IMCU FPCC, respectively, were used for all beakers.Cutting time was in average 30 min.

TABLE 9.1 Cheese making parameters Parameter n s mean Milk, amount in g4000 16 constant GDL in g 3,200 16 constant Milk coagulating FPC 8constant enzyme IMCU/4 FPCC 8 constant CaCl₂/g 1,60 16 Time, min.acidification 30 constant cutting time 2 healing time 3 constantstirring 25 constant scooping 1 constant scooping to press 60 constantpH milk 6.66 setting 6.40 whey n.d Temperature, ° C. setting 31.5Weights, g milk 4000 constant whey 3350 curd 610

Table 9.2 summaries the results on the individual cheese trials, inparticular dry matter (DM) in the whey, with delta-dry-matter calculatedas the difference between FPC amount of whey times DM % deducting FPCCamount of whey times DM %. Average dry a matter was found to be 221.4156g in FPC whey and 221.8666 g in FPCC whey.

TABLE 9.2 Results of the individual cheese trials Dry matter Total dryDelta total Type of in matter in Delta dry dry No. Date rennet Recoverywhey (%) whey matter matter 28.11.00 FPC 98.69 6.639 221.32 1 28.11.00FPCC 98.74 6.606 220.71 0.03304 0.60581 29.11.00 FPC 98.89 6.685 222.142 29.11.00 FPCC 99.00 6.681 222.66 0.00433 −0.51101 30.11.00 FPC 98.386.641 220.62 3 30.11.00 FPCC 98.62 6.602 220.85 0.03898 −0.2235104.12.00 FPC 99.27 6.610 223.60 4 04.12.00 FPCC 98.87 6.628 223.22−0.01807 0.38949 05.12.00 FPC 98.80 6.613 222.36 5 05.12.00 FPCC 98.716.635 222.41 −0.02205 −0.04471 06.12.00 FPC 99.02 6.446 217.65 606.12.00 FPCC 98.95 6.570 221.32 −0.12420 −3.67446 07.12.00 FPC 98.816.365 213.15 7 07.12.00 FPCC 98.61 6.442 214.95 −0.07644 −1.7996512.12.00 FPC 98.81 6.520 218.35 8 12.12.00 FPCC 98.61 6.508 217.150.01279 1.19616 13.12.00 FPC 98.88 6.611 223.18 9 13.12.00 FPCC 98.826.614 222.48 −0.00290 0.70228 15.12.00 FPC 98.78 6.665 222.37 1015.12.00 FPCC 98.87 6.688 223.31 −0.02258 −0.94070 20.12.00 FPC 98.236.685 221.63 11 20.12.00 FPCC 98.26 6.636 219.32 0.04856 2.3135121.12.00 FPC 98.13 6.46 215.13 12 21.12.00 FPCC 98.40 6.50 217.58−0.04364 −2.45507 16.01.01 FPC 98.70 6.752 223.83 13 16.01.01 FPCC 98.736.814 226.32 −0.06126 −2.49389 17.01.01 FPC 98.73 6.801 226.43 1417.01.01 FPCC 98.73 6.747 225.99 0.05351 0.43229 18.01.01 FPC 98.496.695 221.48 15 18.01.01 FPCC 98.63 6.725 222.01 −0.02969 −0.5248009.02.01 FPC 98.88 6.867 229.39 16 09.02.01 FPCC 99.08 6.854 299.580.01354 −0.18775 FPC AVG 6.628 221.4156 −0.0123 −0.4510 FPCC AVG 6.641221.8666 S₂ 0.0467 1.4837

x_(means) on delta-dry-matter was found to be 0.4510, s₂ to be 1.4837and n=16, and thus an estimate for t can be calculated as:t ₀=0.4510/(1.4837/16)_(1/2)=1.4811which shows that the hypothesis that there is no differences between thedry matter losses of FPC and FPCC can be rejected with more than 90%probability.

Speculatively, the difference in dry matter of 0.4510 g could give causeto a cheese of 1 g or a cheese yield increase of 0.16% or 1.6%.

In summary, the sixteen paired laboratory cheese trials, each usingChyMax™ (FPC) and fermented produced camel chymosin (FPCC) were comparedand it was found with more than 90% probability that fermented producedcamel chymosin gives lower dry matter loss to whey, reflecting anexpectation of a higher cheese yield.

REFERENCES

-   Dunn-Coleman, N. S., Bloebaum, P., Berka, R. M., Bodie, E.,    Robinson, N., Armstrong, G., Ward, M., Przetak, M., Carter, G. L.,    LaCost, R., Wilson, L. J., Kodoma, K. H., Baliu, E. F., Houen, G.,    Madsen, M. T, Harlow, K. W., Lønblad, P. and Foltmann, B. (1996) The    Primary Structure and Enzymic Properties of Porcine Prochymosin and    Chymosin, Int. J. Biochem. Cell Biol. 28:667-675.-   Elagamy, E. I. (2000) Physicochemical, molecular and immunological    characterization of camel calf rennet: A comparison with buffalo    rennet, J. Dairy Res. 67:73-81.-   Foltmann, B., Pedersen, V. B., Jacobsen, H., Kauffman, D. and    Wybrandt, G. (1977) The complete amino acid sequence of prochymosin.    Proc. Natl. Acad. Sci. 74:2321-2324.-   Houen, G., Madsen, M. T., Harlow, K. W., Lønblad, P. and    Foltman, b. (1996) The primary structure and enzymatic properties of    porcine prochymosin and chymosin. Int. J. Biochem. Cell. Biol.    28:667-675.-   Kappeler, S. (1998) Compositional and Structural Analysis of Camel    Milk Proteins with Emphasis on Protective Proteins, Dissertation ETH    No. 12947, Swiss Federal Institute of Technology.-   Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular    cloning. A laboratory manual. 2^(nd) edition. Cold Spring Harbor    Laboratory Press.-   Wangoh, J., Farah, Z. and Puhan, Z. (1993). Extraction of camel    rennet and its comparison with calf rennet extract,    Milchwissenschaft 48:322-325.-   Ward, M., Wilson, L. J., Kodoma, K. H., Rey, M. W. and    Berka, R. M. (1990) Improved production of chymosin in Aspergillus    by expression of glycoamylase-chymosin fusion, Bio/Technology    8:435-440.-   Ward, M., Wilson, L. J. and Kodoma, K. H. (1993) Use of Aspergillus    overproducing mutants, cured for integrated plasmid, to overproduce    heterologous proteins, Appl. Microbiol. Biotechnol. 39:738-743.-   WO 94/16086, Chr. Hansen A/S and Bioteknologisk Institut, 21 Jul.    1994

1. An isolated or non-naturally occurring DNA construct, the nucleicacid sequence of which comprises (I) a coding sequence encoding anexpressible protein which is (a) a pre-prochymosin, prochymosin, orchymosin of a mammal of the suborder Tylopoda or (b) a fusion proteincomprising a core protein, wherein said fusion protein is cleavable torelease said core protein and wherein said core protein is apre-prochymosin, prochymosin or chymosin of a mammal of the suborderTylopoda; and (II) appropriate expression signals, operably linked tosaid coding sequence, permitting the protein to be expressed in a hostcell.
 2. The DNA construct of claim 1 in which the mammal is of thegenus Camelus.
 3. The DNA construct of claim 1 in which the mammal isCamelus dromedaries.
 4. An isolated host cell transformed with the DNAconstruct of claim 1, said cell being one in which said expressionsignals are operable.
 5. A method of producing a Tylopoda protein ofinterest selected from the group consisting of pre-prochymosin,prochymosin, and chymosin which comprises providing a host cellaccording to claim 4, cultivating said host cell under conditions wheresaid expressible protein is expressed, if said expressible protein is afusion protein, cleaving it to release said protein of interest, andharvesting the protein of interest.
 6. The method of claim 5 wherein thepre-prochymosin, prochymosin or chymosin is a Camelus dromedariesprotein.
 7. The method of claim 5 wherein the nucleic acid sequenceencodes for a fusion protein comprising pre-prochymosin, prochymosin orchymosin.
 8. The method of claim 7 wherein the fusion protein comprisesglucoamylase or a fragment thereof.
 9. The method of claim 5 wherein thepre-prochymosin, prochymosin, chymosin, or fusion protein is secreted.10. The method of claim 5 wherein the DNA construct comprises pGAMpR.11. The method of claim 5 wherein the DNA construct is pGAMpR-C ascontained in the Aspergillus niger var. awamori strains deposited underthe accession numbers CBS 108915 and CBS
 108916. 12. The method of claim5 wherein the transformed host cell is selected from the groupconsisting of a bacterial cell, a fungal cell, a yeast cell, a mammaliancell, an insect cell and a plant cell.
 13. The method of claim 12wherein the host cell is Aspergillus niger var. awamori.
 14. The methodof claim 13 wherein the Aspergillus niger var. awamori host cell isselected from the group consisting of CBS 108915 and CBS
 108916. 15. Themethod of claim 5 wherein the yield of pre-prochymosin, prochymosin orchymosin milk clotting activity is at least 25% higher than the yield ofbovine pre-prochymosin, bovine prochymosin or bovine chymosin milkclotting activity obtained when using, under identical productionconditions, an expression vector which differs only with respect to itscoding sequence.
 16. The method of claim 5 comprising, as a furtherstep, that the harvested pre-prochymosin, prochymosin or chymosin issubjected to a deglycosylation treatment.
 17. The method of claim 5wherein the host cell is a cell expressing a deglycosylating enzyme. 18.The method of claim 17 wherein the deglycosylating enzyme is endoH. 19.The method of claim 5 in which the mammal is of the genus Camelus.